SDS-PAGE Protein Separation and Identification

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4–12% Criterion XT gradient gels (BioRad, Hercules, CA, USA) with XT MES running buffer (BioRad, Hercules, CA, USA). Gels were run at 125 V for 1 h along with a pre-stained protein standard (Thermo Fisher Scientific, Waltham, MA, USA) and stained for 1.5 h in a 1:1 (v/v) mixture of Coomassie Brilliant Blue R-250 and colloidal Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific, Waltham, MA, USA). After washing in Millipore water overnight to remove excess dye, the stained gels were scanned and analyzed. From selected gel lanes, the protein bands were excised for tryptic digestion [38 (link)] and reconstituted in 50 μL of 1% formic acid in water. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and subsequent data collection were performed as described previously [7 (link)].